Superoxide dismutase-4

ABSTRACT

Polynucleotides which encode the polypeptide SOD-4, as well as such polypeptides, and antibodies against the polypeptide and the use of the polypeptide as a pharmaceutical for treating cerebral ischaemia, ulcers, inflammation, arrhythmia, oedema and paraquat intoxication as well as rheumatoid arthritis, osteoarthritis and radiation injury.

This application is a continuation-in-part of U.S. application Ser. No.08/225,757 filed Apr. 11, 1994 now U.S. Pat. No. 5,506,133.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention is Superoxide Dismutase-4 (SOD-4).

There is a very strong thermodynamic driving force for the reactionsbetween oxygen and biochemical compounds in the body such as proteins,carbohydrates, lipids and nucleic acids. If such reactions go tocompletion, water, carbon dioxide and a number of waste products areformed as end products with the release of large amounts of energy.Oxidation of biological compounds is the source of energy of livingorganisms. Such reactions occur spontaneously but are very slow due toreaction barriers. These barriers are overcome by enzymes inintermediary metabolism, and the final reaction with oxygen takes placein the mitochondria, where the oxygen is reduced by four electrons towater without the liberation of any intermediate products. The reactionis accomplished by cytochrome oxidase complex in the electron transportchain and the energy is bound by the formation of ATP.

However, the direct four step reduction of oxygen to water is unique,and when oxygen reacts spontaneously or is catalyzed by enzymes it isforced to react one step at a time. A series of reactive and toxicintermediates are formed, namely the superoxide radical (O₂ ⁻), hydrogenperoxide (H₂ O₂), and the hydroxyl radical (OH⁻) .

Two of these, O₂ ⁻ and OH⁻, have single unpaired electrons and aretherefore called free radicals. A few percent of the oxygen consumptionin the body has been estimated to lead to the formation of the toxicreduction intermediates. The toxic affects of oxygen are mainlyascribable to the actions of these intermediates.

Oxygen in itself reacts slowly with most biochemical compounds. Thetoxic reactions are in general initiated by processes giving rise tooxygen radicals, which in themselves cause direct damage to biochemicalcompounds or start chain reactions involving oxygen.

Some compounds react spontaneously with oxygen, i.e., they autoxidize.Virtually all autoxidations result in the formation of toxic oxygenreduction intermediates. Autoxidation of adrenalin, pyrogallol andseveral other compounds lead to the formation of the superoxide radical.When ionizing radiation passes through an aqueous solution containingoxygen, the superoxide radical is the radical found in the highestconcentration. The toxic oxygen reduction products so formed are offundamental importance for the killing ability of the cells, but mayalso lead to damage in the surrounding tissue.

Hydrogen peroxide is always formed when superoxide is formed by way ofthe dismutation reaction. Most oxidases in the body directly reduceoxygen to hydrogen peroxide.

Organisms living in the presence of oxygen have been forced to develop anumber of protective mechanisms against the toxic oxygen reductionmetabolites. The protective factors include superoxide dismutases (SOD)which dismutate the superoxide radical and are found in relativelyconstant amounts in mammalian cells and tissue. The best known of theseenzymes is CuZnSOD which is a dimer with a molecular weight of 33,000containing two copper and two zinc atoms. CuZnSOD is found in thecytosol and in the intermembrane space of the mitochondria. MnSOD is atetramer with a molecular weight of 85,000 containing four Mn atoms, andis mainly located in the mitochondrial matrix. Until recently the extracellular fluids were assumed to lack SOD activity. However U.S. Pat. No.5,248,603 recently disclosed the presence of a superoxide dismutase inextracellular fluids (e.g., blood plasma, lymph, synovial fluid andcerebrospinal fluid) which was termed EC-SOD.

Crystallographic structures of recombinant human CuZnSOD have beendetermined, refined and analyzed at 2.5 A resolution for wild-type and adesigned thermal stable double-mutant enzyme (Cys-6--Ala, Cys-111--Ser).There is a helix dipole interaction with a Zn site, and 14 residues formtwo or more structurally conserved side-chain to main-chain hydrogenbonds that appear critical to active-site architecture, loopconfirmation and the increased stability resulting from the Cys-111--Sermutation. Parge, H. E. et al, Proc. Natl. Acad. Sci. U.S.A., 89:6109-13(1992).

Mutations in the CuZnSOD gene occur in patients with the fatalneurodegenerative disorder familial amyotrophic lateral sclerosis.Screening of the CuZnSOD coding region revealed that the mutation Ala 4to Val in exon 1 was the most frequent one, mutations were identified inexons 2, 4 and 5 but not in the active site region formed by exon 3.Thus, defective CuZnSOD is linked to motor neuron death and carriesimplications for understanding and possible treatment of familialamyotrophic lateral sclerosis. The polypeptide of the present invention,SOD-4, is structurally and functionally related to CuZnSOD.

Japanese Patent No. 4327541 discloses a therapeutic drug forimmuno-reactions with organs after transplantation containing the activesubstance of human CuZnSOD obtained by gene recombination.

Japanese Patent No. 4312533 discloses a composition for treatingcerebral ischaemia which comprises recombinant Cuzn human SOD andinhibits delayed nerve necrosis accompanying is chaemia.

Japanese Patent No. 4248984 discloses a superoxide dismutase derivativewhich has a longer half-life in blood than SOD and therefore helps treatvarious diseases.

European Patent No. 499621 discloses a method for purifying recombinantCuZnSOD and a method for increasing the yield of the B isoform analog ofthis polypeptide.

Japanese Patent No. 2156884 discloses a 153 amino acid polypeptidehaving human superoxide dismutase properties and a DNA sequence encodingsuch polypeptide, a DNA sequence expressed by the nucleic acid sequenceand production of the polypeptide by culture of host cells.

Japanese Patent No. 63313581. discloses a pharmacologically activemodified superoxide dismutase which is obtained by reacting SOD with acompound containing an amino or carboxyl group.

Japanese Patent No. 63077822 discloses an agent for improving thefunction of organs which uses a human SOD-like polypeptide as the activesubstance.

In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide which is SOD-4, as well asfragments, analogs and derivatives thereof. The polypeptide of thepresent invention is of human origin.

In accordance with another aspect of the present invention, there areprovided polynucleotides (DNA or RNA) which encode such polypeptides.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptides by recombinanttechniques.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides for therapeutic purposes, forexample, for treating inflammatory pathologies, ulcers, arrhythmia,ischaemia, oedema, paraquat intoxication, rheumatoid arthritis andosteoarthritis, reducing reperfusion injuries and decreasing bloodpressure.

In accordance with yet a further aspect of the present invention, thereis provided an antibody against such polypeptides. These and otheraspects of the present invention should be apparent to those skilled inthe art from the teachings herein.

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 shows the cDNA sequence (SEQ ID NO:1) and deduced amino acidsequence for the (SEQ ID NO:2) SOD-4 gene. The amino acid sequenceencodes for one of the mature forms of the polypeptide, since there areat least two in-frame ATG start codons. The mature polypeptide couldstart at either one of the ATG codons. The standard one letterabbreviation for amino acids is used.

FIG. 2 displays the amino acid homology between SOD-4 (SEQ ID NO:2) witheleven other cytosolic CuZnSODs from various species. Schistosome (SEQID NO:3), Bovine (SEQ ID NO:4), Cauliflower (SEQ ID NO:5), Droposphila(SEQ ID NO:6), Human SOD1 (SEQ ID NO:7), Tomato (SEQ ID NO:8), Maize(SEQ ID NO:9), Mouse (SEQ ID NO:10), Xenopus (SEQ ID NO:11), and S.cerevisiae (SEQ ID NO:12). The copper-zinc-bind-sites (in boldface type)are formed by six His residues and one Asp residue. The Arg (R) residueis believed necessary to guide the superoxide to the activity site.Identical residues are represented by dashes and deletions arerepresented by dots.

FIG. 3 shows the results of bacterial expression and purification ofhuman SOD-4 after separation on an SDS polyacrylamide gel.

FIG. 4 shows the results of expression of recombinant SOD-4 in COS cellsafter separation on an SDS polyacrylamide gel.

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIG. 1 or for themature polypeptide encoded by the cDNA of the clone deposited as ATCCDeposit No. 75716 on Mar. 22, 1994.

The polynucleotide of the present invention was isolated from an earlystage human brain cDNA library. It contains an open reading frameencoding a polypeptide of 255 amino acids. The polypeptide has thehighest degree of homology to CuZnSOD isolated from Schistosoma mansonihaving 51% identity and 72% similarity over a 151 amino acid overlap.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 or that of the deposited clone ormay be a different coding sequence which coding sequence, as a result ofthe redundancy or degeneracy of the genetic code, encodes the same,mature polypeptide as the DNA of FIG. 1 or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIG. 1 orfor the mature polypeptide encoded by the deposited cDNA may include:only the coding sequence for the mature polypeptide; the coding sequencefor the mature polypeptide and additional coding sequence such as aleader or secretory sequence or a proprotein sequence; the codingsequence for the mature polypeptide (and optionally additional codingsequence) and non-coding sequence, such as introns or non-codingsequence 5' and/or 3' of the coding sequence for the mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 or the polypeptide encoded by the cDNA of the deposited clone.The variant of the polynucleotide may be a naturally occurring allelicvariant of the polynucleotide or a non-naturally occurring variant ofthe polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1 or the same mature polypeptideencoded by the cDNA of the deposited clone as well as variants of suchpolynucleotides which variants encode for a fragment, derivative oranalog of the polypeptide of FIG. 1 or the polypeptide encoded by thecDNA of the deposited clone. Such nucleotide variants include deletionvariants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 or of the coding sequence of the deposited clone. Asknown in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexahistidine tag supplied by a pD10 vector to providefor purification of the mature polypeptide fused to the marker in thecase of a bacterial host, or, for example, the marker sequence may be ahemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 50% andpreferably 70% identity between the sequences. The present inventionparticularly relates to polynucleotides which hybridize under stringentconditions to the hereinabove-described polynucleotides. As herein used,the term stringent conditions, means hybridization will occur only ifthere is at least 95% and preferably at least 97% identity between thesequences. The polynucleotides which hybridize to the hereinabovedescribed polynucleotides in a preferred embodiment encode polypeptideswhich retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNA of FIG. 1 or the depositedcDNA.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

The present invention further relates to a SOD-4 polypeptide which hasthe deduced amino acid sequence of FIG. 1 or which has the amino acidsequence encoded by the deposited cDNA, as well as fragments, analogsand derivatives of such polypeptide.

The terms "fragment," "derivative" and "analog" when referring to thepolypeptide of FIG. 1 or that encoded by the deposited cDNA, means apolypeptide which retains essentially the same biological function oractivity as such polypeptide. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 or thatencoded by the deposited cDNA may be (i) one in which one or more of theamino acid residues are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or (ii) one in which one or more of the amino acidresidues includes a substituent group, or (iii) one in which the maturepolypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused tothe mature polypeptide, such as a leader or secretory sequence or asequence which is employed for purification of the mature polypeptide ora proprotein sequence. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art from theteachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term "isolated" means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered, (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the SOD-4 genes. The culture conditions,such as temperature, pH and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomvces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila and Sf9;animal cells such as CHO, COS or Bowes melanoma; plant cells, etc. Theselection of an appropriate host is deemed to be within the scope ofthose skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pRK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation. (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the pblypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 "backbone" sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5' flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The polypeptides can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatogradhy hydroxylapatite chromatography and lectinchromatography. It is preferred to have low concentrations(approximately 0.15-5 mM) of calcium ion present during purification.(Price et al., J. Biol. Chem., 244:917 (1969)). Protein refolding stepscan be used, as necessary, in completing configuration of the matureprotein. Finally, high performance liquid chromatography (HPLC) can beemployed for final purification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

SOD-4 may also be employed as an anti-inflammatory agent. Other SODproteins have been shown to exhibit an anti-inflammatory affect in aseries of animal models of inflammation as well as in inflammatorydiseases in animals (Huber et al, eds. Michelson el al, Academic Press,517-549, (1977). SOD-4 may also be used to treat rheumatoid arthritisand the adverse effects of ionizing radiation since, in humans, positiveaffects have been shown using SOD proteins to treat rheumatoid arthritisand arthroses as well as adverse affects of treatment with ionizingradiation. The mechanism by which SOD-4 works is by removing oxidationproducts, which products cause tissue degeneration.

SOD-4 may be used to treat Crohn's disease, Bechet's disease,dermatitis, ulcers, ulcerative colitis, and against the adverse effectsof radiation therapy. Other SOD proteins have been found to be effectiveagainst these conditions (Niwa, Y et al, Free Rad. Res. Comms. 1:137-153(1985)).

If the supply of blood to a tissue is cut off, the tissue will slowlybecome necrotic. Oxygen radicals formed as a result of the reappearanceof oxygen in previously ischaemic tissue appear to contribute to thedamage. Thus the removal of these free radicals by SOD-4 helps toprotect tissue against damage. SOD-4 may be employed to reduce theincidence of ischaemia and reperfusion induced arrhythmias by a similarmechanism, since SOD proteins have been reported to affect theseconditions (Woodward, B. et al, J. Mol. Cell. Cardiol. 17:485-493(1985). In the same manner, SOD-4 may be employed to treat cerebralischaemia and kidney ischaemia, SOD proteins have been demonstrated toprotect tissues in ischaemia or anoxiareperfusion models in the kidney(Baker, G. L., et al., Am. Surg., 202:628-41 (1985).

Also, SOD-4 may be employed in connection with kidney transplantationsand other organ transplantations such as skin, lung, liver and pancreas.

SOD-4 may be employed to treat burns. The local oedema after anexperimental slight burn in rats could be somewhat decreased throughinjection of SOD proteins (Bjork and Artursson, Burns, 9:249-256 (1983).

Parenterally administered CuZnSOD has been reported to preventbronchopulmonary dysplasia in preterm neonates suffering from infantilerespiratory distress. The CuZnSOD has recently received orphan drugstatus for this treatment. Accordingly, SOD-4 may also be employed totreat these diseases also. (Rosenfeld W. et al, J. Pediatr. 105:781-785(1984).

In various types of autoimmune diseases, such as systemic lupuserythematosus, and rheumatoid arthritis an increased frequency ofchromosomal breaks in lymphocytes has been demonstrated. Plasma fromsuch patients contains a chromosome breaking factor, called clastogenicfactor. Superoxide radicals in the plasma results in formation of thisfactor. SOD-4 may protect against this clastogenic activity by removingthe superoxide radicals.

Superoxide radicals tend to damage cells, DNA and proteins by oxidativestress which may disrupt the normal cell cycle and lead to uncontrolleddivision of cells which is the basis of a cancer. Accordingly, SOD-4 canbe employed to prevent or control cancer by the removal of superoxideradicals from a patient's system.

Oxygen radicals contribute to the damaging affects of a number of toxicsubstances such as paraquat and alloxan. SOD-4 may protect against thesetoxic substances through direct injection.

Alloxan has been reported to have diabetogenic activity. SOD-4 mayprotect against this diabetogenic activity of alloxan in vivo.Beta-cells of the pancreas are extremely sensitive to alloxan, and thissensitivity may lead to insulin-dependent diabetes mellitus. It maytherefore be contemplated to protect the Beta cells with injections withSOD-4 at the first onset of diabetes mellitus.

The polypeptides may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as "gene therapy."

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

The polypeptides of the present invention may be employed in combinationwith a suitable pharmaceutical carrier. Such compositions comprise atherapeutically effective amount of the polypeptide, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention may be employed in conjunctionwith other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. Theamounts and dosage regimens of SOD-4 administered to a subject willdepend on a number of factors such as the mode of administration, thenature of the condition being treated and the judgment of theprescribing physician. Generally speaking, they are given, for example,in therapeutically effective doses of at least about 10 μg/kg bodyweight and in most cases they will be administered in an amount not inexcess of about 8 mg/Kg body weight per day and preferably the dosage isfrom about 10 μg/kg to about 1 mg/kg body weight daily, taking intoaccount the routes of administration, symptoms, etc.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the primer will yield an amplifiedfragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clones to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 500 or 600 bases; however, clones larger than 2,000 bp havea higher likelihood of binding to a unique chromosomal location withsufficient signal intensity for simple detection. FISH requires use ofthe clones from which the EST was derived, and the longer the better.For example, 2,000 bp is good, 4,000 is better, and more than 4,000 isprobably not necessary to get good results a reasonable percentage ofthe time. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that cDNA sequence. Ultimately, completesequencing of genes from several individuals is required to confirm thepresence of a mutation and to distinguish mutations from polymorphisms.

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention.

In accordance with a further aspect of the present invention, there isprovided a process for determining susceptibility to disorders directlyrelated to a mutation in the SOD-4 gene product. Such disorders includebut are not limited, amyotrophic lateral sclerosis "ALS", andParkinson's disease. Thus, a mutation in an SOD-4 protein indicates asusceptibility to these disorders, and the nucleic acid sequencesencoding an SOD-4 polypeptide may be employed in an assay forascertaining such susceptibility. Thus, for example, the assay may beemployed to determine a mutation in a human SOD-4 protein as hereindescribed, such as a deletion, truncation, insertion, frameship, etc.,with such mutation being indicative of a susceptibility to theabove-mentioned diseases.

A mutation may be ascertained, for example, by a DNA sequencing assay.Tissue samples, including but not limited to blood samples, are obtainedfrom a patient. The samples are processed by methods known in the art tocapture the RNA. The first strand cDNA is synthesized from the RNAsamples by adding an oligonucleotide primer consisting of polythymidineresidues which hybridize to the polyadenosine stretch present on themRNA's. Reverse transcriptase and deoxynucleotides are added to allowsynthesis of the first strand cDNA. Primer sequences are synthesizedbased on the DNA sequence of the SOD-4 polypeptide of the invention. Theprimer sequence is generally comprised of 15 to 30 and preferable from18 to 25 consecutive basis of the SOD-4 gene. The primers are used inpairs (one "sense" and one "anti-sense") to amplify the cDNA from thepatients by the PCR method such that three overlapping fragments of thepatients' cDNAs are generated. The overlapping fragments are thensubjected to dideoxynucleotide sequencing using a set of primersequences synthesized to correspond to the base pairs of the cDNAs at apoint approximately every 200 base pairs throughout the gene. The primersequences are used for sequencing to determine where a mutation in thepatients' SOD-4 protein may be. The sequence information determined fromthe patient is then compared to non-mutated sequences to determine ifany mutations are present.

In another embodiment, the primer sequences are used in the PCR methodto amplify a mutated region. The region could be sequenced and used as adiagnostic tool to predict a predisposition to such mutated genes.

Alternatively, the assay to detect mutations in the SOD-4 gene may beperformed by generating cDNA from the RNA and expressing the proteinencoded by the cDNA by in vitro transcription and translation. Theexpressed protein may then be analyzed by electrophoresis on an SDS,polyacrylamide or other gel. A "normal" SOD-4 gene product is alsoelectrophoresed on the gel, and the gel is then dried and subjected toauto-radiography and the suspected mutated gene product and the "normal"gene product are analyzed and any differences in the banding pattern ofsuch gene products are indicative of a mutation in the cDNA. A mutationin the gene product can also be detected by using SOD-4 antibody in aWestern Blot analysis. Accordingly, the mutations in the genes of thepresent invention may be determined directly by sequencing or indirectlyby examining an expressed protein.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

"Oligonucleotides" refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5' phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units to T4 DNA ligase ("ligase")per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A.., Virology, 52:456-457(1973).

EXAMPLE 1

Bacterial Expression and Purification of SOD-4

The DNA sequence encoding for SOD-4, ATCC # 75716 is initially amplifiedusing PCR oligonucleotide primers corresponding to the 5' and 3'sequences of the processed SOD-4 protein (minus the signal peptidesequence) and the vector sequences 3' to the SOD-4 gene. Additionalnucleotides corresponding to SOD-4 are added to the 5' and 3' sequencesrespectively. The 5' oligonucleotide primer has the sequence 5'-CGGGATCCATGGGCAGCGGCCAGTTG-3' and (SEQ ID NO:13) contains a Bam HIrestriction enzyme site followed by 18 nucleotides of SOD-4 codingsequence starting from one of the presumed terminal amino acids of theprocessed protein. The 3' sequence, 5'-CGTCTAGAGGTCCTGCTCAAAGGTGGG-3'(SEQ ID NO:14) contains complementary sequences to an Xba I restrictionsite and the last 21 nucleotides of SOD-4 and to a pD10 vector sequencelocated 3' to the SOD-4 DNA insert. The restriction enzyme sitescorrespond to the restriction enzyme sites on the bacterial expressionvector pD10 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, Calif. 91311).pD10 encodes antibiotic resistance (Amp'), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator. (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pD10 was then digested with Bam HI and Xba I. The amplified sequenceswere ligated into pD10 and were inserted in frame with the sequenceencoding for the histidine tag and the RBS. The ligation mixture wasthen used to transform E. coli strain M15/rep4 available from Qiagenunder the trademark M15/rep 4 by the procedure described in Sambrook, Jet al., Molecular Cloning: A Laboratory Manual, Cold Spring LaboratoryPress, 1989. M15/rep4 contains multiple copies of the plasmid pREP4,which expresses the lacI repressor and also confers kanamycin resistance(Kan'). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies were selected.Plasmid DNA was isolated and confirmed by restriction analysis. Clonescontaining the desired constructs were grown overnight (O/N) in liquidculture in LB media supplemented with both Amp (100 μg/ml) and Kan (25μg/ml). The O/N culture is used to inoculate a large culture at a ratioof 1:100 to 1:250. The cells were grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalactopyranoside") was then added to a final concentration of 1 mM. IPTGinduces by inactivating the lacI repressor, clearing the P/O leading toincreased gene expression. Cells were grown an extra 3 to 4 hours. Cellswere then harvested by centrifugation. The cell pellet was solubilizedin the chaotropic agent 6 Molar Guanidine HCl. After clarification,solubilized SOD-4 was purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag. Hochuli, E. et al., J. Chromatography411:177-184 (1984). Proteins from different stages of purification wereseparated on a 12.5% SDS polyacrylamide gel and stained with Coomassieblue dye. M represents a molecular sizing marker. Lanes 1 and 2 aretotal extracts from bacteria containing the vector pD10 in the absence(lane 1) and presence (lane 2) of IPTG. Lanes 3 and 4 are total extractsfrom bacteria containing the expression plasmid pD10-SOD-4 in theabsence (lane 3) and presence (lane 4) of IPTG. Lanes 5 through 9represent elution fractions from a Nickel-Chelate column. Lane 5 isflow-through; lanes 6 and 7 represent elution fractions washed with 6 Mguanidine HCl, 50 mM NaPO₄, pH 8 and pH 6; lanes 8 and 9 are elutionfractions washed with 6 M guanidine HCl 50 mM NaPO, pH 5 and pH 2. SeeFIG. 3.

EXAMPLE 2

Expression of Recombinant SOD-4 in COS cells

The expression of plasmid, pSOD-4-HA is -derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E. coli replication origin, 4) CMVpromoter followed by a polylinker region, a SV40 intron andpolyadenylation site. A DNA fragment encoding the entire SOD-4 precursorand a HA tag fused in frame to its 3' end was cloned into the polylinkerregion of the vector, therefore, the recombinant protein expression isdirected under the CMV promoter. The HA tag correspond to an epitopederived from the influenza hemagglutinin protein as previously described(I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R.Lerner, 1984, Cell 37, 767). The infusion of HA tag to our targetprotein allows easy detection of the recombinant protein with anantibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding for SOD-4, ATCC # 75716 was constructed by PCRusing two primers: the 5' primer sequence 5'-AATTAACCCTCACTAAAGGG-3' in(SEQ ID NO:15) pBluescript vector; the 3' sequence5'-CGCTCTAGACAAGCGTAGCTGGGACGTCGTATGGGTGGTGGGCA GGGGGCTG-3' (SEQ IDNO:16) contains complementary sequences to an Xba I restriction enzymesite, translation stop codon, HA tag and the last 18 nucleotides of theSOD-4 coding sequence (not including the stop codon). Therefore, the PCRproduct contains a Bam HI site from the pBluescript vector, SOD-4 codingsequence followed by HA tag fused in frame, a translation terminationstop codon next to the HA tag, and an Xba I site. The PCR amplified DNAfragment and the vector, pbluescript, were digested with Bam HI and XbaI restriction enzymes and ligated. The ligation mixture was transformedinto E. coli strain SURE (available from Stratagene Cloning Systems,11099 North Torrey Pines Road, La Jolla, Calif. 92037) the transformedculture was plated on ampicillin media plates and resistant colonieswere selected. Plasmid DNA was isolated from transformants and examinedby restriction analysis for the presence of the correct fragment. Forexpression of the recombinant SOD-4, COS cells were transfected with theexpression vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T.Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring LaboratoryPress, (1989)). The expression of the SOD-4-HA protein was detected byradiolabelling and immunoprecipitation method. (E. Harlow, D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)). Proteins were labelled for 8 hours with ³⁵ S-cysteine two dayspost transfection. Culture media were then collected and cells werelysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM Tris, pH 7.5). (Wilson, I. et al., Id. 37:767(1984)). ³⁵ S-cysteine labeled proteins from COS cell lysates andsupernatants were immunoprecipitated with an HA polyclonal antibody andseparated using 15% SDS-PAGE. M equals molecular weight markers. Lanes 1through 4 are cell lysates. Lanes 5 through 8 are supernatants. Lanes 1and 5 are mock controls with no DNA. Lanes 2 and 6 are MIP-1γ controlfor secreted proteins. Lanes 3 and 7 are control for cell lysate andlanes 4 and 8 are SOD-4. See FIG. 4.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 16    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1080 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (cDNA)    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 115..879    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CTGGTTGGTGCTCCTGCGCCGGAGGAGTTCTGCGTCTCGGGGTGGTGACTGGGTCCAGAA60    TGGCTTCGGATTGGGGAACAGGGGACCCTCTGCACGTTGGAGTTCGCGGTGCAGATG117    Met    ACCTGTCAGAGCTGTGTGGACGCGGTGCGCAAATCCCTGCAAGGGGTG165    ThrCysGlnSerCysValAspAlaValArgLysSerLeuGlnGlyVal    51015    GCAGGTGTCCAGGATGTGGAGGTGCACTTGGAGGACCAGATGGTCTTG213    AlaGlyValGlnAspValGluValHisLeuGluAspGlnMetValLeu    202530    GTACACACCACTCTACCCAGCCAGGAGGTGCAGGCTCTCCTGGAAGGC261    ValHisThrThrLeuProSerGlnGluValGlnAlaLeuLeuGluGly    354045    ACGGGGCGGCAGGCGGTACTCAAGGGCATGGGCAGCGGCCAGTTGCAG309    ThrGlyArgGlnAlaValLeuLysGlyMetGlySerGlyGlnLeuGln    50556065    AATCTGGGGGCAGCAGTGGCCATCCTGGGGGGGGCTGGCACCGTGCAG357    AsnLeuGlyAlaAlaValAlaIleLeuGlyGlyAlaGlyThrValGln    707580    GGGGTGGTGCGCTTCCTACAGCTGACCCCTGAGCGCTGCCTCATCGAG405    GlyValValArgPheLeuGlnLeuThrProGluArgCysLeuIleGlu    859095    GGAACTATTGACGGCCTGGAGCCTGGGCTGCATGGACTCCACGTCCAT453    GlyThrIleAspGlyLeuGluProGlyLeuHisGlyLeuHisValHis    100105110    CAGTACGGGGACCTTACAAACAACTGCAACAGCTGTGGGAATCACTTT501    GlnTyrGlyAspLeuThrAsnAsnCysAsnSerCysGlyAsnHisPhe    115120125    AACCCTGATGGAGCATCTCATGGGGGCCCCCAGGACTCTGACCGGCAC549    AsnProAspGlyAlaSerHisGlyGlyProGlnAspSerAspArgHis    130135140145    CGCGGAGACCTGGGCAATGTCCGTGCTGATGCTGACGGCCGCGCCATC597    ArgGlyAspLeuGlyAsnValArgAlaAspAlaAspGlyArgAlaIle    150155160    TTCAGAATGGAGGATGAGCAGCTGAAGGTGTGGGATGTGATTGCCCGC645    PheArgMetGluAspGluGlnLeuLysValTrpAspValIleAlaArg    165170175    AGCCTGATTATTGATGAGGGAGAAGATGACCTGGGCCGGGGAGGCCAT693    SerLeuIleIleAspGluGlyGluAspAspLeuGlyArgGlyGlyHis    180185190    CCCTTATCCAAGATCACAGGGAACTCCGGGGAGAGGTTGGCCTGTGGC741    ProLeuSerLysIleThrGlyAsnSerGlyGluArgLeuAlaCysGly    195200205    ATCATTGCACGCTCCGCTGGCCTTTTCCAGAACCCCAAGCAGATCTGC789    IleIleAlaArgSerAlaGlyLeuPheGlnAsnProLysGlnIleCys    210215220225    TCTTGCGATGGCCTCACCATCTGGGAGGAGCGAGGCCGGCCCATCGCT837    SerCysAspGlyLeuThrIleTrpGluGluArgGlyArgProIleAla    230235240    GGCAAGGGCCGAAAGGAGTCAGCGCAGCCCCCTGCCCACCTT879    GlyLysGlyArgLysGluSerAlaGlnProProAlaHisLeu    245250255    TGAGCAGGACCTCACCTTGGCTCTGTTGCTGTCCTCCAGGGCGAGCACTTTCCACTTCCA939    GAGGGGGCCAGAGGGACTTTGCCTGCCCAGTCTTTGGAGAGCTCAGTACAGGGCAGGAGC999    TGCTGTGGTGTTCCCTTGGCAAATGAAAGTTTTATTTTCGTTTGGGAAAAAAAAAAAAAA1059    AAAAAAAAAAAAAAAAAAAAA1080    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 255 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetThrCysGlnSerCysValAspAlaValArgLysSerLeuGlnGly    151015    ValAlaGlyValGlnAspValGluValHisLeuGluAspGlnMetVal    202530    LeuValHisThrThrLeuProSerGlnGluValGlnAlaLeuLeuGlu    354045    GlyThrGlyArgGlnAlaValLeuLysGlyMetGlySerGlyGlnLeu    505560    GlnAsnLeuGlyAlaAlaValAlaIleLeuGlyGlyAlaGlyThrVal    65707580    GlnGlyValValArgPheLeuGlnLeuThrProGluArgCysLeuIle    859095    GluGlyThrIleAspGlyLeuGluProGlyLeuHisGlyLeuHisVal    100105110    HisGlnTyrGlyAspLeuThrAsnAsnCysAsnSerCysGlyAsnHis    115120125    PheAsnProAspGlyAlaSerHisGlyGlyProGlnAspSerAspArg    130135140    HisArgGlyAspLeuGlyAsnValArgAlaAspAlaAspGlyArgAla    145150155160    IlePheArgMetGluAspGluGlnLeuLysValTrpAspValIleAla    165170175    ArgSerLeuIleIleAspGluGlyGluAspAspLeuGlyArgGlyGly    180185190    HisProLeuSerLysIleThrGlyAsnSerGlyGluArgLeuAlaCys    195200205    GlyIleIleAlaArgSerAlaGlyLeuPheGlnAsnProLysGlnIle    210215220    CysSerCysAspGlyLeuThrIleTrpGluGluArgGlyArgProIle    225230235240    AlaGlyLysGlyArgLysGluSerAlaGlnProProAlaHisLeu    245250255    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 153 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    MetLysAlaValCysValMetThrGlyThrAlaGlyValLysGlyVal    151015    ValLysPheThrGlnGluThrAspAsnGlyProValHisValHisAla    202530    GluPheSerGlyLeuLysAlaGlyLysHisGlyPheHisValHisGlu    354045    PheGlyAspThrThrAsnGlyCysThrSerAlaGlyAlaHisPheAsn    505560    ProThrLysGlnGluHisGlyAlaProGluAspSerIleArgHisVal    65707580    GlyAspLeuGlyAsnValValAlaGlyAlaAspGlyAsnAlaValTyr    859095    AsnAlaThrAspLysLeuIleSerLeuAsnGlySerHisSerIleIle    100105110    GlyArgSerMetValIleHisGluAsnGluAspAspLeuGlyArgGly    115120125    GlyHisGluLeuSerLysValThrGlyAsnAlaGlyGlyArgLeuAla    130135140    CysGlyValValGlyLeuAlaAlaGlu    145150    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 150 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    ThrLysAlaValCysValLeuLysGlyAspGlyProValGlnGlyThr    151015    IleHisPheGluAlaLysGlyAspThrValValValThrGlySerIle    202530    ThrGlyLeuThrGluGlyAspHisGlyPheHisValHisGlnPheGly    354045    AspAsnThrGlnGlyCysThrSerAlaGlyProHisPheAsnProLeu    505560    SerLysLysHisGlyGlyProLysAspGluGluArgHisValGlyAsp    65707580    LeuGlyAsnValThrAlaAspLysAsnGlyValAlaIleValAspIle    859095    ValAspProLeuIleSerLeuSerGlyGluTyrSerIleIleGlyArg    100105110    ThrMetValValHisGluLysProAspAspLeuGlyArgGlyGlyAsn    115120125    GluGluSerThrLysThrGlyAsnAlaGlySerArgLeuAlaCysGly    130135140    ValIleGlyIleIleLys    145150    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 151 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    AlaLysGlyValCysValLeuAsnSerSerGluGlyValLysGlyThr    151015    IlePhePheThrHisGluGlyAsnGlyAlaThrThrValThrGlyThr    202530    ValSerGlyLeuArgProGlyLeuHisGlyPheHisValHisAlaLeu    354045    GlyAspAsnThrAsnGlyCysMetSerThrGlyProHisPheAsnPro    505560    AspGlyLysThrHisGlyAlaProGluAspAlaAsnArgHisAlaGly    65707580    AspLeuGlyAsnIleIleValGlyAspAspGlyThrAlaThrPheThr    859095    IleThrAspSerGlnIleProLeuSerGlyProAsnSerIleValGly    100105110    ArgAlaIleValValHisAlaAspProAspAspLeuGlyLysGlyGly    115120125    HisGluLeuSerLeuSerThrGlyAsnAlaGlyGlyArgValAlaCys    130135140    GlyIleIleGlyIleGlnGly    145150    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 151 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    ValLysAlaValCysValIleAsnGlyAspAlaLysGlyThrValPhe    151015    PheGluGlnGluSerSerGlyThrProValLysValSerGlyGluVal    202530    CysGlyLeuAlaLysGlyLeuHisGlyPheHisValHisGluPheGly    354045    AspAsnThrAsnGlyCysMetSerSerGlyProHisPheAsnProTyr    505560    GlyLysGluHisGlyAlaProValAspGluAsnArgHisLeuGlyAsp    65707580    LeuGlyAsnIleGluAlaThrGlyAspCysProThrLysValAsnIle    859095    ThrAspSerLysIleThrLeuPheGlyAlaAspSerIleIleGlyArg    100105110    ThrValValValHisAlaAspAlaAspAspLeuGlyGlnGlyGlyHis    115120125    GluLeuSerLysSerThrGlyAsnAlaGlyAlaArgIleGlyCysGly    130135140    ValIleGlyIleIleLysVal    145150    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 152 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    ThrLysAlaValCysValLeuLysGlyAspGlyProValGlnGlyIle    151015    IleAsnPheGluGlnLysGluSerAsnGlyProValLysValTrpGly    202530    SerIleLysGlyLeuThrGluGlyLeuHisGlyPheHisValHisGlu    354045    PheGlyAspAsnThrAlaGlyCysThrSerAlaGlyProHisPheAsn    505560    ProLeuSerArgLysHisGlyGlyProLysAspGluGluArgHisVal    65707580    GlyAspLeuGlyAsnValThrAlaAspLysAspGlyValAlaAspVal    859095    SerIleGluAspSerValIleSerLeuSerGlyAspHisCysIleIle    100105110    GlyArgThrLeuValValHisGluLysAlaAspAspLeuGlyLysGly    115120125    GlyAsnGluGluSerThrLysThrGlyAsnAlaGlySerArgLeuAla    130135140    CysGlyValIleGlyIleIleGln    145150    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 151 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    ValLysAlaValCysValLeuAsnSerSerGluGlyValSerGlyThr    151015    TyrLeuPheThrGlnValGlyValAlaProThrThrValAsnGlyAsn    202530    IleSerGlyLeuLysProGlyLeuHisGlyPheHisValHisAlaLeu    354045    GlyAspAsnThrAsnGlyCysMetSerThrGlyProHisTyrAsnPro    505560    AlaGlyLysGluHisGlyAlaProGluAspGluValArgHisValGly    65707580    AspLeuGlyAsnIleThrValGlyGluAspGlyThrAlaSerPheThr    859095    IleThrAspLysGlnIleProLeuThrGlyProGlnSerIleIleGly    100105110    ArgAlaValValValHisAlaAspProAspAspLeuGlyLysGlyGly    115120125    HisGluLeuSerLysSerThrGlyAsnAlaGlyGlyArgIleAlaCys    130135140    GlyIleIleGlyIleGlnGly    145150    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 150 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    ValLysAlaValAlaValLeuAlaGlyThrAspValLysGlyThrIle    151015    PhePheSerGlnGluGlyAspGlyProThrThrValThrGlySerIle    202530    SerGlyLeuLysProGlyLeuHisGlyPheHisValHisAlaLeuGly    354045    AspThrThrAsnGlyCysMetSerThrGlyProHisPheAsnProVal    505560    GlyLysGluHisGlyAlaProGluAspGluAspArgHisAlaGlyAsp    65707580    LeuGlyAsnValThrAlaGlyGluAspGlyValValAsnValAsnIle    859095    ThrAspSerGlnIleProLeuAlaGlyProHisSerIleIleGlyArg    100105110    AlaValValValHisAlaAspProAspAspLeuGlyLysGlyGlyHis    115120125    GluLeuSerLysSerThrGlyAsnAlaGlyGlyArgValAlaCysGly    130135140    IleIleGlyIleGlnGly    145150    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 151 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    MetLysAlaValCysValLeuLysGlyAspGlyProValGlnGlyThr    151015    IleHisPheGluGlnLysAlaSerGlyGluProTrpLeuSerGlyGln    202530    IleThrGlyLeuThrGluGlyGlnHisGlyPheHisValHisGlnTyr    354045    GlyAspAsnThrGlnGlyCysThrSerAlaGlyProHisPheAsnPro    505560    HisSerLysLysHisGlyGlyProAlaAspGluGluArgHisValGly    65707580    AspLeuGlyAsnValThrAlaGlyLysAspGlyValAlaAsnValSer    859095    IleGluAspArgValIleSerLeuSerGlyGluHisSerIleIleGly    100105110    ArgThrMetValValHisGluLysGlnAspAspLeuGlyLysGlyGly    115120125    AsnGluGluSerThrLysThrGlyAsnAlaGlySerArgLeuAlaCys    130135140    GlyValIleGlyIleIleGln    145150    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 150 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    ValLysAlaValCysValLeuAlaGlySerGlyAspValLysGlyVal    151015    ValArgPheGluGlnGlnAspAspGlyAspValThrValGluGlyLys    202530    IleGluGlyLeuThrAspGlyAsnHisGlyPheHisIleHisValPhe    354045    GlyAspAsnThrAsnGlyCysLeuSerAlaGlyProHisPheAsnPro    505560    GlnAsnLysAsnHisGlySerProLysAspAlaAspArgHisValGly    65707580    AspLeuGlyAsnValThrAlaGluGlyGlyValAlaGlnPheAsnPhe    859095    ThrAspProGlnIleSerLeuLysGlyGluArgSerIleIleGlyArg    100105110    ThrAlaValValHisGluLysGlnAspAspLeuGlyLysGlyGlyAsp    115120125    AspGluSerLeuLysThrGlyAsnAlaGlyGlyArgLeuAlaCysGly    130135140    ValIleGlyPheCysPro    145150    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 152 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    ValGlnAlaValAlaValLeuLysGlyAspAlaGlyValSerGlyVal    151015    ValLysPheGluGlnAlaSerGluSerGluProThrThrValSerTyr    202530    GluIleAlaGlyAsnSerProAsnAlaGluArgPheHisIleHisGlu    354045    PheGlyAspAlaThrAsnGlyCysValSerAlaGlyProHisPheAsn    505560    ProPheLysLysThrHisGlyAlaProThrAspGluValArgHisVal    65707580    GlyAspMetGlyAsnValLysThrAspGluAsnGlyValAlaLysGly    859095    SerPheLysAspSerLeuIleLysLeuIleGlyProThrSerValVal    100105110    GlyArgSerValValIleHisAlaGlyGlnAspAspLeuGlyLysGly    115120125    AspThrGluGluSerLeuLysThrGlyAsnAlaGlyProArgProAla    130135140    CysGlyValIleGlyIleThrAsn    145150    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (cDNA)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    CGGGATCCATGGGCAGCGGCCAGTTG26    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (cDNA)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    CGTCTAGAGGTCCTGCTCAAAGGTGGG27    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (cDNA)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    AATTAACCCTCACTAAAGGG20    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 57 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (cDNA)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAAAGGTGGGCAGGGGGCTG57    __________________________________________________________________________

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of:(i) a SOD-4 polypeptide having the deduced amino acidsequence of amino acids 1 to 255 of FIG. 1 (SEQ ID NO:2) and fragmentsthereof; (ii) a SOD-4 polypeptide having the amino acid sequence ofamino acids 2 to 255 of FIG. 1 (SEO ID NO:2) and fragments thereof; and(iii) a SOD-4 polypeptide encoded by the cDNA of ATCC Deposit No. 75716and fragments thereof.
 2. The polypeptide of claim 1 wherein thepolypeptide is SOD-4 having the deduced amino acid sequence of aminoacids I to 255 of FIG. 1 (SEQ ID NO:2).
 3. The polypeptide of claim 1,wherein the polypeptide has the amino acid sequence of amino acids 2 to255 of FIG. 1 (SEQ ID NO:2).
 4. The polypeptide of claim 1, wherein thepolypeptide has the amino acid sequence as encoded by the cDNA of ATCCDeposit No.
 75716. 5. A pharmaceutical composition comprising thepolypeptide of claim 1 and a pharmaceutically acceptable carrier.
 6. Amethod for the treatment of a patient having need of SOD-4 comprising:administering to the patient a therapeutically effective amount of thepolypeptide of claim 1.